Mixing Of Aqueous Contents Research Articles

Bacterial dynamin-like protein DynA mediates lipid and content mixing.

Many bacterial species contain dynamin-like proteins (DLPs). However, so far the functional mechanisms of bacterial DLPs are poorly understood. DynA in Bacillus subtilis is a 2-headed DLP, mediating nucleotide-independent membrane tethering in vitro and contributing to the innate immunity of bacteria against membrane stress and phage infection. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if DynA induces membrane full fusion, characterize its subunits in membrane fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer indicated that DynA could induce aqueous content mixing even in the absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow process, and it has phospholipid and membrane curvature preferences. The D1 part of DynA is crucial for membrane binding and fusion, whereas D2 subunit plays a role in facilitating membrane fusion. Surprisingly, digestion of DynA mediated an instant rise of content exchange, supporting the assumption that disassembly of DynA is a driving force for fusion-through-hemifusion. DynA is a rare example of a membrane fusion catalyst that lacks a transmembrane domain and hence sets this system apart from well-characterized fusion systems such as the soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes.-Guo, L., Bramkamp, M. Bacterial dynamin-like protein DynA mediates lipid and content mixing.

Biophysical Studies of LC3 Family Proteins.

Autophagy is an important cellular process in which cell components are degraded in a controlled way and their building blocks are recycled into new macromolecules. Autophagy starts within a double-membrane container, the autophagosome, itself the result of a number of interconversions of cell membranous elements. In our recent work, we have described reconstituted model systems for the interactions of autophagy proteins with membrane lipid bilayers and for the autophagy protein-mediated vesicle tethering and fusion, with the aim of ultimately reconstituting the autophagosome formation. The present chapter describes in detail (a) the steps required for the preparation of semisynthetic lipid vesicles (liposomes), including giant unilamellar vesicles, (b) ultracentrifugation and fluorescence methods for assaying protein binding to membranes, and (c) procedures for assessing vesicle-vesicle aggregation and fusion. The latter include methods for intervesicular total lipid mixing, mixing of lipids in the vesicle inner monolayers, and aqueous contents mixing.

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR 2, a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR 2 is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR 2 when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR 2 cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR 2 on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR 2-induced liposome aggregation, as long as Ca 2+ is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis < vesicle aggregation < total lipid mixing < inner lipid mixing < contents mixing. Concomitant measurements of the threshold diacylglyceride + ceramide concentrations in the bilayer show that late events, e.g. lipid mixing, require a higher concentration of PlcHR 2 products than early ones, e.g. aggregation. When the above results are examined in the context of the membrane effects of other phospholipid phosphocholine hydrolases it can be concluded that aggregation is necessary, but not sufficient for membrane fusion to occur, that diacylglycerol is far more fusogenic than ceramide, and that vesicle membrane permeabilization occurs independently from vesicle fusion.

Reversible, cooperative reactions of yeast vacuole docking

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block. Using them, we test whether docking entails a linearly ordered series of catalytic events, marked by sequential acquisition of resistance to inhibitors, or whether docking subreactions are cooperative and/or reversible. We find that each fusion protein and regulatory lipid is needed throughout docking, indicative of a reversible or highly cooperative assembly of the fusion-competent vertex ring. In accord with this cooperativity, vertices enriched in one fusion catalyst are enriched in others. Docked vacuoles finally assemble SNARE complexes, yet still require physiological temperature and lipid rearrangements to complete fusion.

Membrane fusion induced by the catalytic activity of a phospholipase C/sphingomyelinase from Listeria monocytogenes.

Listeria monocytogenes is a bacterium responsible for localized and generalized infections in humans and animals. It has the ability to spread from the cytoplasm of an infected cell to neighboring cells without becoming exposed to the extracellular space. The bacterium secretes a phospholipase C (PLC(LM)) that is active on glycerophospholipids, e.g., phosphatidylcholine, and on sphingomyelin; thus, PLC(LM) should be described more appropriately as a phospholipase C/sphingomyelinase. We have obtained PLC(LM) free from a frequent contaminant, listeriolysin O, using an improved purification procedure. PLC(LM) has been assayed on large unilamellar liposomes of defined lipid composition. The enzyme is activated by K(+) and Mg(2+), and readily degrades phospholipids in bilayer form, in the absence of detergents. Enzyme activity is accompanied by important changes in the structure of the phospholipid vesicles, namely, vesicle aggregation, intervesicular mixing of lipids, and mixing of aqueous contents, with very low leakage of vesicular contents. The data are interpreted as indicative of PLC(LM)-induced vesicle fusion. This is confirmed by the demonstration of intervesicular mixing of inner monolayer lipids, using a novel procedure. The observation of PLC(LM)-induced membrane fusion suggests a mechanism for the cell-to-cell propagation of the bacterium, which requires disruption of a double-membrane vacuole.

A soluble peripherin/Rds C-terminal polypeptide promotes membrane fusion and changes conformation upon membrane association

Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311–325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284–346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS–PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS–PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial α-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in α-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in α-helical content.

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers.

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection.

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.

The paramyxovirus fusion protein forms an extremely stable core trimer: structural parallels to influenza virus haemagglutinin and HIV-1 gp41.

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit F2 and a membrane anchored subunit F1. A highly stable structure has been identified comprised of peptides derived from the simian virus 5 (SV5) F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the SV5 F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm > 90 degrees C), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 M(r)) that is resistant to denaturation by 2% SDS at 40 degrees C. The authors suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that is either fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptide N-1 inhibits cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41 and influenza virus haemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.

A Core Trimer of the Paramyxovirus Fusion Protein: Parallels to Influenza Virus Hemagglutinin and HIV-1 gp41

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit, F2, and a membrane-anchored subunit, F1. We have identified a highly stable structure composed of peptides derived from the F1heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the F1heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% α-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm>90°C), 82% α-helical, discrete trimer of heterodimers (mass 31,300Mr) that is resistant to denaturation by 2% SDS at 40°C. We suggest that this α-helical trimeric complex represents the core most stable form of the F protein that either is fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptides N-1 and N-2 inhibit cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41, and influenza virus hemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.

Dual inhibitory effect of gangliosides on phospholipase C-promoted fusion of lipidic vesicles.

The effect of a variety of gangliosides has been tested on the phospholipase C-induced fusion of large unilamellar vesicles. Bilayer composition was phosphatidylcholine:phosphatidylethanolamine: cholesterol (2:1:1 mole ratio) plus the appropriate amounts of glycosphingolipids. Enzyme phosphohydrolase activity, vesicle aggregation, mixing of bilayer lipids and mixing of liposomal aqueous contents were separately assayed. Small amounts ( < 1 mol %) of gangliosides in the lipid bilayer produce a significant inhibition of the above processes. The inhibitory effect of gangliosides increases with the size of the oligosaccharide chain in the polar head group. Inhibition depends in a nonlinear manner on the ganglioside proportion, and is complete at approximately 5 mol %. Inhibition is not due to ganglioside-dependent changes in vesicle curvature or size. Ganglioside inhibition of vesicle fusion is due to two different effects: inhibition of phospholipase C activity and stabilization of the lipid lamellar phase. Enzyme inhibition leads to a parallel decrease of vesicle aggregation and lipid mixing rates. Mixing of aqueous contents, though, is depressed beyond the enzyme inhibition levels. This is explained in terms of the fusion pore requiring a local destabilization of the lipid bilayer, the lamellar structure being stabilized by gangliosides. 31P-NMR and DSC experiments confirm the inhibitory effect of gangliosides in various lamellar-to-nonlamellar transitions.

Topological properties of two cubic phases of a phospholipid : cholesterol: diacylglycerol aqueous system and their possible implications in the phospholipase C-induced liposome fusion

Water dispersions of phospholipid: cholesterol: diacylglycerol may, under certain conditions, originate either the lipid- and water-permeable Q 224 cubic phase, or the lipid-permeable but water-impermeable Q 227 cubic phase. These results are discussed within the framework of the phospholipase C-induced fusion of liposomes [Nieva et al. (1993) Biochemistry 32, 1054]. It is suggested that the cubic phases Q 224 and Q 227 represnt two classes of lipid organization, one promoting, the other hindering the mixing of aqueous contents that is characteristic of membrane fusion. In this context, inverted micelles appear to be the end point of the fusion process, rather than fusion intermediates.

In Vitro Fusion of Reticulocyte Endocytic Vesicles with Liposomes

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Interaction of stearylamine-liposomes with erythrocyte ghosts: analysis of membrane lipid mixing and aqueous contents mixing, and the effect of carboxymethyl chitin on the interaction

We have investigated the interaction of stearylamine (SA)-containing liposomes (SA-liposomes) with erythrocyte ghosts (EGs), utilizing the Tb-dipicolinate (Tb-DPA) assay for the mixing of the aqueous contents and a resonance energy transfer (RET) assay for the mixing of lipid. The results demonstrate that SA-liposomes and EGs, after aggregation, have a great tendency to mix their lipid before the mixing of the internal contents. The mixing of their contents takes place inside the vesicles due to the fusion of SA-liposomes and EGs, followed by the leakage of the contents from the vesicles. We have also explored the properties of the interaction of SA-liposomes with carboxymethyl chitin (CM-chitin), an inhibitor of the lipid mixing between SA-liposomes and EGs, using fluorescein-labeled CM-chitin (Flu-CMC). The polarization value of Flu-CMC increased upon SA-liposome binding, the amount of Flu-CMC on the liposome surfaces increased with the increase in the concentration of phospholipid and SA, and it decreased by 20–40% in the presence of 10 mM Ca 2+ ions, which have a high affinity for CM-chitin. These results show that the association of CM-chitin with SA-liposomes could be due to both electrostatic and hydrophobic interactions between SA-liposomes and CM-chitin.

Mechanistic Study on Toxicity of Positively Charged Liposomes Containing Stearylamine to Blood

We studied the interaction of stearylamine (SA) containing liposomes (SA-liposomes) with erythrocyte ghost (EG) or platelets, utilizing the Tb/dipicolinate (Tb/DPA) assay for the mixing of aqueous contents and a resonance energy transfer (RET) assay for the mixing of lipid. The results demonstrate that SA-liposomes and EG, after aggregation, have a great tendency to mix their lipid before the mixing of the internal contents. The mixing of their contents takes place inside the vesicles due to the fusion of SA-liposomes and EG, followed by the leakage of the contents from the vesicles. In the presence of carboxymethyl chitin (CM-chitin), SA-liposomes-EG interaction was inhibited, indicating that CM-chitin reduces the tendency of SA-liposomes to interact with EG. The lipid mixing between SA-liposomes and platelets was not affected by CM-chitin or phagocytosis inhibitors: EDTA, cytochalasin B, or 2,4-dinitrophenol and iodoacetate, indicating the importance of glycoproteins on the platelet membrane surface in the interaction of SA-liposomes with platelets.

Capacities of pardaxin analogues to induce fusion and leakage of negatively charged phospholipid vesicles are not necessarily correlated.

Peptide-induced vesicle fusion is frequently accompanied by leakage of vesicle contents. To determine the correlation between these two processes, we studied the interaction of the amphiphilic peptide pardaxin and two of its analogues with large unilamellar vesicles composed of phosphatidylserine. A pardaxin analogue with a positive charge at both its C- and N-termini induced significantly more fusion but less leakage than the parent peptide. Fusion and leakage were studied with large unilamellar vesicles of two sizes. Aggregation of vesicles was found to be the rate-limiting step in the overall fusion process induced by the peptides. The rates and extents of fusion, determined by membrane mixing, increase in vesicle size, and mixing of aqueous contents, were significantly enhanced in the presence of 2.5-5 mM Mg2+ which promoted vesicle aggregation. Model calculations showed that increasing the peptide to lipid ratio resulted in a parallel increase in the fusion rate constants. As the average vesicle diameter was increased, the extent of leakage was enhanced, as more peptide molecules were bound to each vesicle. The mode of leakage induced by the peptides was also investigated. Our results suggest that the potency of a peptide to induce vesicle fusion is not necessarily associated with its capacity to induce leakage, and we further elucidate how these capacities depend on the structures of the peptides.

Time-resolved and equilibrium measurements of the effects of poly(ethylene glycol) on small unilamellar phospholipid vesicles.

The effects of poly(ethylene glycol) (PEG) on sonicated unilamellar vesicles made of phosphatidylcholine have been examined. Stopped-flow and equilibrium data are presented for vesicle aggregation, vesicle leakage, lipid mixing, and aqueous contents mixing. Vesicle aggregation is detected as a monoexponential increase in light scattering, for PEG concentrations between 5 and 10%. In the region between 10-15% and 23-27% PEG, under our experimental conditions, the increase in light scattering follows a more complex biexponential kinetics, and, under these conditions, vesicle aggregation is accompanied by lipid mixing, a combination of events denoted as "close apposition". Above 23-27% PEG, the increase in light scattering is accompanied by fast lipid mixing, and also mixing of aqueous contents, all this being indicative of vesicle fusion; in addition, leakage occurs under these circumstances. Fusion takes place at high PEG concentrations, as indicated above, without any dilution step. From a methodological point of view, the Tb/DPA assay is shown to be more appropriate than the ANTS/DPX method for leakage and fusion studies in the presence of PEG.

Phospholipase C-promoted membrane fusion. Retroinhibition by the end-product diacylglycerol.

The catalytic activity of phospholipase C induces fusion of pure lipid vesicles. When large unilamellar liposomes composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) are treated with phospholipase C, in the presence of 10 mM Ca2+, two enzyme effects can be distinguished: a fast one (half-time on the order of seconds) consisting mainly of vesicle-vesicle fusion and a slow one (half-time on the order of minutes) representing bulk lipid hydrolysis. The fast fusion process is inhibited by the end-product diacylglycerol, as well as by lysophosphatidylcholine and by low Ca2+ concentrations. The temperature dependence of enzyme activity (phospholipid hydrolysis), vesicle aggregation, and vesicle fusion (mixing of aqueous contents) has been separately studied. Enzyme activity and vesicle aggregation rates increase monotonically with temperature, while an optimum temperature is found for vesicle fusion, depending on liposome composition and assay conditions. The presence of diacylglycerol incorporated to the membrane (up to 10 mol %) does not produce any fusion effect even at temperatures as high as 80 degrees C: in situ diacylglycerol production by the enzyme appears to be required. The data are interpreted in support of a hypothesis according to which a "fusion intermediate" would be required, depending (among others) on bilayer composition, temperature, and Ca2+ concentration, for vesicle fusion to occur.

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.